The present invention relates to the use of inhibitors of stem cell proliferation for regulating stem cell cycle in the treatment of humans or animals having autoimmune diseases, aging, cancer, myelodysplasia, preleukemia, leukemia, psoriasis or other diseases involving hyperproliferative conditions. The present invention also relates to a method of treatment for humans or animals anticipating or having undergone exposure to chemotherapeutic agents, other agents which damage cycling stem cells, or radiation exposure. Finally, the present invention relates to the improvement of the stem cell maintenance or expansion cultures for auto- and allo-transplantation procedures or for gene transfer.
Most end-stage cells in renewing systems are short-lived and must be replaced continuously throughout life. For example, blood cells originate from a self-renewing population of multipotent hematopoietic stem cells (HSC). Hematopoietic stem cells are a subpopulation of hematopoietic cells. Hematopoietic cells can be obtained, for example, from bone marrow, umbilical cord blood and peripheral blood (either unmobilized or mobilized with an agent such as G-CSF); hematopoietic cells include the stem cell population, progenitor cells, differentiated cells, accessory cells, stromal cells and other cells that contribute to the environment necessary for production of mature blood cells. Because the hematopoietic stem cells are necessary for the development of all of the mature cells of the hematopoietic and immune systems, their survival is essential in order to reestablish a fully functional host defense system in subjects treated with chemotherapy or other agents.
Hematopoietic cell production is regulated by a series of factors that stimulate growth and differentiation of hematopoietic cells, some of which, for example erythropoietin and G-CSF, are currently used in clinical practice. One part of the control network which has not been extensively characterized, however, is the feedback mechanism that forms the negative arm of the regulatory process (Eaves et al. Blood 78:110-117, 1991).
Early studies by Lord and coworkers showed the existence of a soluble protein factor in normal murine and porcine bone marrow extracts, which was capable of reversibly inhibiting the cycling of HSC (Lord et al., Br. J. Haem. 34:441-446, 1976). This inhibitory activity (50-100 kD molecular weight) was designated stem cell inhibitor (SCI).
Purification of this factor from primary sources was not accomplished due to the difficulties inherent in an in vivo assay requiring large numbers of irradiated mice. In an attempt to overcome these problems Pragnell and co-workers developed an in vitro assay for primitive hematopoietic cells (CFU-A) and screened cell lines as a source of the inhibitory activity (see Graham et al. Nature 344:442-444, 1990).
As earlier studies had identified macrophages as possible sources for SCI (Lord et al. Blood Cells 6:581-593, 1980), a mouse macrophage cell line, J7742, was selected (Graham et al. Nature 344:442-444, 1990). The conditioned medium from this cell line was used by Graham et al. for purification; an inhibitory peptide was isolated which proved to be identical to the previously described cytokine macrophage inflammatory protein 1-alpha (MIP-1xcex1). Thus, MIP-1xcex1 was isolated from a cell line, not from primary material. While Graham et al., observed that antibody to MIP-1xcex1 abrogated the activity of a crude bone marrow extract, other workers have shown that other inhibitory activities are important. For example, Graham et al. (J. Exp. Med. 178:925-32, 1993) have suggested that TGFxcex2, not MIP-1xcex1, is a primary inhibitor of hematopoietic stem cells. Further, Eaves et al. (PNAS 90:12015-19, 1993) have suggested that both MIP-1xcex1 and TGFxcex2 are present at sub optimal levels in normal bone marrow and that inhibition requires a synergy between the two factors.
Other workers have described additional stem cell inhibitory factors. Frindel and coworkers have isolated a tetrapeptide from fetal calf marrow and from liver extracts which has stem cell inhibitory activities (Lenfant et al., PNAS 86:779-782, 1989). Paukovits et al. (Cancer Res. 50:328-332, 1990) have characterized a pentapeptide which, in its monomeric form, is an inhibitor and, in its dimeric form, is a stimulator of stem cell cycling. Other factors have also been claimed to be inhibitory in various in vitro systems (see Wright and Pragneil in Bailliere""s Clinical Haematology v. 5, pp. 723-39, 1992 (Bailliere Tinadail, Paris)).
Tsyrlova et al., SU 1561261 A1, disclosed a purification process for a stem cell proliferation inhibitor.
To date, none of these factors have been approved for clinical use. However, the need exists for effective stem cell inhibitors. The major toxicity associated with chemotherapy or radiation treatment is the destruction of normal proliferating cells which can result in bone marrow suppression or gastrointestinal toxicity. An effective stem cell inhibitor would protect these cells and allow for the optimization of these therapeutic regimens. Just as there is a proven need for a variety of stimulatory cytokines (i.e., cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-14, IL-15, G-CSF, GM-CSF, erythropoietin, thrombopoietin, stem cell factor, flk2/flt3 ligand, etc., which stimulate the cycling of hematopoietic cells) depending upon the clinical situation, so too it is likely that a variety of inhibitory factors will be needed to address divergent clinical needs.
Hemoglobin is a highly conserved tetrameric protein with molecular weight of approximately 64,000 Daltons. It consists of two alpha and two beta chains. Each chain binds a single molecule of heme (ferroprotoporphyrin IX), an iron-containing prosthetic group. Vertebrate alpha and beta chains were probably derived from a single ancestral gene which duplicated and then diverged; the two chains retain a large degree of sequence identity both between themselves and between various vertebrates (see FIG. 16A). In humans, the alpha chain cluster on chromosome 16 contains two alpha genes (alpha1 and alpha2) which code for identical polypeptides, as well as genes coding for other alpha-like chains: zeta, theta and several non-transcribed pseudogenes (see FIG. 16B for cDNA and amino acid sequences of human alpha chain). The beta chain cluster on chromosome 11 consists of one beta chain gene and several beta-like genes: delta, epsilon, G gamma and A gamma, as well as at least two unexpressed pseudogenes (see FIG. 16C for cDNA and amino acid sequences of human beta chain).
The expression of these genes varies during development. In human hematopoiesis, which has been extensively characterized, embryonic erythroblasts successively synthesize tetramers of two zeta chains and two epsilon chains (Gower I), two alpha chains and two epsilon chains (Gower II) or two zeta chains and two gamma chains (Hb Portland). As embryogenesis proceeds, the predominant form consists of fetal hemoglobin (Hb F) which is composed of two alpha chains and two gamma chains. Adult hemoglobin (two alpha and two beta chains) begins to be synthesized during the fetal period: at birth approximately 50% of hemoglobin is of the adult form and the transition is complete by about 6 months of age. The vast majority of hemoglobin (approximately 97%) in the adult is of the two alpha and two beta chain variety (Hb A) with small amounts of Hb F or of delta chain (Hb A2) being detectable.
Heme has been extensively examined with regard to its influences on hematopoiesis (see S. Sassa, Seminars Hemat. 25:312-20, 1988 and N. Abraham et al., Int. J. Cell Cloning 9:185-210, 1991 for reviews). Heme is required for the maturation of erythroblasts; in vitro, hemin (chloroferroprotoporphyrin IXxe2x80x94i.e., heme with an additional chloride ion) increases the proliferation of CFU-GEMM, BEU-E and CFU-E. Similarly, hemin increases cellularity in long-term bone marrow cultures.
I. Chemotherapy and Radiotherapy of Cancer
Productive research on stimulatory growth factors has resulted in the clinical use of number of these factors (erythropoietin, G-CSF, GM-CSF, etc.). These factors have reduced the mortality and morbidity associated with chemotherapeutic and radiation treatments. Further clinical benefits to patients who are undergoing chemotherapy or radiation could be realized by an alternative strategy of blocking entrance of stem cells into cell cycle thereby protecting them from toxic side effects.
II. Bone Marrow Transplantation
Bone marrow transplantation (BMT) is a useful treatment for a variety of hematological, autoimmune and malignant diseases; current therapies include hematopoietic cells obtained from umbilical cord blood or from peripheral blood (either unmobilized or mobilized with agents such as G-CSF) as well as from bone marrow. Ex vivo manipulation of hematopoietic cells is currently being used to expand primitive stem cells to a population suitable for transplantation. Optimization of this procedure requires: (1) sufficient numbers of stem cells able to maintain long term reconstitution of hematopoiesis; (2) the depletion of graft versus host-inducing T-lymphocytes and (3) the absence of residual malignant cells. This procedure can be optimized by including a stem cell inhibitor(s) for ex vivo expansion.
The effectiveness of purging of hematopoietic cells with cytotoxic drugs in order to eliminate residual malignant cells is limited due to the toxicity of these compounds for normal hematopoietic cells and especially stem cells. There is a need for effective protection of normal cells during purging: protection can be afforded by taking stem cells out of cycle with an effective inhibitor.
III. Peripheral Stem Cell Harvesting
Peripheral blood stem cells (PBSC) offer a number of potential advantages over bone marrow for autologous transplantation. Patients without suitable marrow harvest sites due to tumor involvement or previous radiotherapy can still undergo PBSC collections. The use of blood stem cells eliminates the need for general anesthesia and a surgical procedure in patients who would not tolerate this well. The apheresis technology necessary to collect blood cells is efficient and widely available at most major medical centers. The major limitations of the method are both the low normal steady state frequency of stem cells in peripheral blood and their high cycle status after mobilization procedures with drugs or growth factors (e.g., cyclophosphamide, G-CSF, stem cell factor). An effective stem cell inhibitor would be useful to return such cells to a quiescent state, thereby preventing their loss through differentiation.
IV. Treatment of Hyperproliferative Disorders
A number of diseases are characterized by a hyperproliferative state in which dysregulated stem cells give rise to an overproduction of end stage cells. Such disease states include, but are not restricted to, psoriasis, in which there is an overproduction of epidermal cells, and premalignant conditions in the gastrointestinal tract characterized by the appearance of intestinal polyps. A stem cell inhibitor would be useful in the treatment of such conditions.
V. Gene Transfer
The ability to transfer genetic information into hematopoietic cells is currently being utilized in clinical settings. Hematopoietic cells are a useful target for gene therapy because of ease of access, extensive experience in manipulating and treating this tissue ex vivo and because of the ability of blood cells to permeate tissues. Furthermore, the correction of certain human genetic defects may be possible by the insertion of a functional gene into the primitive stem cells of the human hematopoietic system.
There are several limitations for the introduction of genes into human hematopoietic cells using either retrovirus vectors or physical techniques of gene transfer: (1) The low frequency of stem cells in hematopoietic tissues has necessitated the development of high efficiency gene transfer techniques; and (2) more rapidly cycling stem cells proved to be more susceptible to vector infection, but the increase of the infection frequency by stimulation of stem cell proliferation with growth factors produces negative effects on long term gene expression, because cells containing the transgenes are forced to differentiate irreversibly and lose their self-renewal. These problems can be ameliorated by the use of a stem cell inhibitor to prevent differentiation and loss of self-renewal.
The present invention relates to polypeptides which are inhibitors of stem cell proliferation (xe2x80x9cINPROLxe2x80x9d) and their use.
The present invention includes an inhibitor of stem cell proliferation characterized by the following properties:
(a) Specific activity (IC50) less than or equal to 20 ng/ml in a murine colony-forming spleen (CFU-S) assay (see Example 4),
(b) Molecular weight greater than 10,000 and less than 100,000 daltons (by ultrafiltration),
(c) Activity sensitive to degradation by trypsin,
(d) More hydrophobic than MIP-1xcex1 or TGFxcex2 and separable from both by reverse phase chromatography (see Example 12),
(e) Biological activity retained after heating for one hour at 37xc2x0 C., 55xc2x0 C. or 75xc2x0 C. in aqueous solution and
(f) Biological activity retained after precipitation with 1% hydrochloric acid in acetone.
The present invention is further characterized and distinguished from other candidate stem cell inhibitors (e.g., MIP-1xcex1, TGFxcex2 and various oligopeptides) by its capacity to achieve inhibition in an in vitro assay after a short preincubation period (see Example 5).
The present invention also comprises pharmaceutical compositions containing INPROL for treatment of a variety of disorders.
The present invention provides a method of treating a subject anticipating exposure to an agent capable of killing or damaging stem cells by administering to that subject an effective amount of a stem cell inhibitory composition. The stem cells protected by this method may be hematopoietic stem cells ordinarily present and dividing in the bone marrow. Alternatively, stem cells may be epithelial, located for example, in the intestines or scalp or other areas of the body or germ cells located in reproductive organs. The method of this invention may be desirably employed on humans, although animal treatment is also encompassed by this method. As used herein, the terms xe2x80x9csubjectxe2x80x9d or xe2x80x9cpatientxe2x80x9d refer to an animal, such as a mammal, including a human.
In another aspect, the invention provides a method for protecting and restoring the hematopoietic, immune or other stem cell systems of a patient undergoing chemotherapy, which includes administering to the patient an effective amount of INPROL.
In still a further aspect, the present invention involves a method for adjunctively treating any cancer, including those characterized by solid tumors, by administering to a patient having cancer an effective amount of INPROL to protect stem cells of the bone marrow, gastrointestinal tract or other organs from the toxic effects of chemotherapy or radiation therapy.
Yet another aspect of the present invention involves the treatment of leukemia, comprising treating hematopoietic cells having proliferating leukemia cells therein with an effective amount of INPROL to inhibit proliferation of normal stem cells, and treating the bone marrow with a cytotoxic agent to destroy leukemia cells. This method may be enhanced by the follow-up treatment of the bone marrow with other agents that stimulate its proliferation: e.g., colony stimulating factors. In one embodiment this method is performed in vivo. Alternatively, this method is also useful for ex vivo purging and expansion of hematopoietic cells for transplantation.
In still a further aspect, the method involves treating a subject having any disorder caused by proliferating stem cells. Such disorders, such as psoriasis, myelodysplasia, some autoimmune diseases, immuno-depression in aging, are treated by administering to the subject an effective amount of INPROL to partially inhibit proliferation of the stem cell in question.
The present invention provides a method for reversibly protecting stem cells from damage from a cytotoxic agent capable of killing or damaging stem cells. The method involves administering to a subject anticipating exposure to such an agent an effective amount of INPROL.
The present invention also provides:
An inhibitor of stem cell proliferation isolated from porcine or other bone marrow by the following procedure (see Example 12):
(a) Extraction of bone marrow and removal of particulate matter through filtration,
(b) Heat treatment at 56xc2x0 C. for 40 minutes followed by cooling in ice bath,
(c) Removal of precipitate by centrifugation at 10,000 g for 30 minutes at 4xc2x0 C.,
(d) Acid precipitation by addition of supernatant to 10 volumes of stirred ice-cold acetone containing 1% by volume concentrated hydrochloric acid and incubation at 4xc2x0 C. for 16 hours.
(e) Isolation of precipitate by centrifugation at 20,000 g for 30 minutes at 4xc2x0 C. and washing with cold acetone followed by drying,
(f) Isolation by reverse phase chromatography and monitoring activity by inhibition of colony formation by bone marrow cells pretreated with 5-fluorouracil and incubated in the presence of murine IL-3, as well as by absorption at 280 nm and by SDS-PAGE.
The present invention also provides:
A method for purifying an inhibitor of stem cell proliferation substantially free from other proteinaceous materials comprising the preceding steps, as also described in more detail below.
The present invention also provides:
A method of treatment for humans or animals wherein an inhibitor of stem cell proliferation functions to ameliorate immunosuppression caused by stem cell hyperproliferation.
The present invention also provides:
A method of treatment for humans or animals wherein said inhibitor of stem cell proliferation is administered after the stem cells are induced to proliferate by exposure to a cytotoxic drug or irradiation procedure. Stem cells are normally quiescent but are stimulated to enter cell cycle after chemotherapy. This renders them more sensitive to a second administration of chemotherapy; the current method protects them from this treatment.
The present invention also provides:
A method of treatment for humans or animals wherein said inhibitor of stem cell proliferation is administered as an adjuvant before or together with vaccination for the purpose of increasing immune response.
The present invention also provides:
A method of treatment for humans or animals receiving cytotoxic drugs or radiation treatment which comprises administering an effective amount of the inhibitor of stem cell proliferation to protect stem cells against damage.
The invention also includes a pharmaceutical composition comprising hemoglobin and a pharmaceutically acceptible carrier.
The invention also includes a pharmaceutical composition comprising (a) a polypeptide selected from the group consisting of the alpha chain of hemoglobin, the beta chain of hemoglobin, the gamma chain of hemoglobin, the delta chain of hemoglobin, the epsilon chain of hemoglobin and the zeta chain of hemoglobin, and (b) a pharmaceutically acceptible carrier. Such pharmaceutical compositions can composed of a single polypeptide selected from said group, a mixture of polypeptides selected from said group or polypeptides from said group in the form of dimers or multimers, with or without heme.
The invention also includes peptides having the sequences:
Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val, (SEQ ID NO:1)
Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ ID NO:2)
where the two Cys residues form a disulfide bond,
Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (SEQ ID NO:2)
where the two Cys residues are joined by a carbon bridge, and
Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala (SEQ ID NO:3).
Also included in the invention are DNA sequences encoding the above identified peptides, vectors containing said DNA sequences and host cells containing said vectors. These peptides can by synthesized using standard chemical techniques (e.g., solid phase synthesis) or by using recombinant techniques (e.g., fusion systems such as those employing glutathione-S-transferase (D. B. Smith and K. S. Johnson, Gene 67:31-40, 1988), thioredoxin (LaVallie et al., Biotechnology 11:187-193, 1993) or ubiquitin (Butt et al., PNAS 86:2540-4, 1989; U.S. Pat. No. 5,391,490)).
Additionally the invention includes a method of inhibiting stem cell proliferation comprising contacting hematopoietic cells with a compound capable of binding opiate receptors, advantageously the mu subclass of opiate receptors. Peptides (called xe2x80x9chemorphinsxe2x80x9d) have been isolated from hemoglobin which exhibit opiate-like activities (e.g., Brantl et al., Eur. J. Pharm, 125:309-10, 1986; Davis et al. Peptides 10:747-51, 1989; Karelin et al. Bioch. Biophys. Res. Comm, 202:410-5, 1994; Zhao et al., Ann. N.Y. Acad. Sci 750:452-8, 1995). Each of these articles is hereby incorporated by reference.
The invention also includes a method of inhibiting stem cell proliferation comprising contacting hematopoietic cells with a peptide selected from the group of hemorphin peptides having the sequence:
Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:4),
Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:5),
Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:6),
Leu-Val-Val-Tyr-Pro-Trp-Thr (SEQ ID NO:7),
Leu-Val-Val-Tyr-Pro-Trp (SEQ ID NO:8),
Leu-Val-Val-Tyr-Pro (SEQ ID NO:9),
Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:10),
Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:11),
Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:12),
Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:13), and
Tyr-Pro-Trp-Thr (SEQ ID NO:14).
The above peptides have sequence similarity to other opiate-like peptides such as those of the Tyr-MIF-1 family (see Reed et al., Neurosci. Biobehav. Rev. 18:519-25, 1994 for review), the casein-derived casomorphins (Brantl et al., Hoppe-Seyler""s Z. Physiol. Chem. 360:1211-16, 1979; Loukas et al., Biochem. 22:4567-4573, 1983; Fiat and Jolles, Mol. Cell. Biochem. 87:5-30, 1989), peptides derived from cytochrome b, termed cytochrophins (Brantl et al., Eur. J. Pharm. 111:293-4, 1985) as well as peptides derived from combinatorial libraries screened for binding to opiate receptors (see Dooley et al., Peptide Research 8:124-137, 1995 for review). Each of these articles is hereby incorporated by reference.
The invention also includes a method of inhibiting stem cell proliferation comprising contacting hematopoietic cells with a peptide selected from the group consisting of Tyr-MIF-1 related peptides, casomorphins and cytochrophins. Specifically included are the Tyr-MIF-1 peptides having the sequences:
Tyr-Pro-Trp-Gly-NH2,
Tyr-Pro-Lys-Gly-NH2,
Tyr-Pro-Leu-Gly-NH2, and
Pro-Leu-Gly-NH2.
The invention also includes a method of conducting gene therapy in a mammal comprising:
a) removing hematopoietic cells from said mammal.
b) optionally treating said hematopoietic cells ex vivo with at least one stimulatory cytokine to induce stem cell proliferation,
c) transfecting said hematopoietic cells with a pre-determined gene,
d) contacting said transfected hematopoietic cells ex vivo with INPROL,
e) transplanting into a mammal the hematopoietic cells of step d,
f) optionally treating said mammal in vivo with INPROL.
The invention also includes a method of conducting ex vivo stem cell expansion comprising treating said hematopoietic cells with INPROL and at least on stimulatory cytokine. INPROL is contacted with the hematopoietic cells before, during and/or after contact with the stimulatory cytokine.
The invention also includes a pharmaceutical composition comprising (a) INPROL and (b) at least one inhibitory compound selected from the group consisting of MIP-1xcex1, TGFxcex2, TNFxcex1, INFxcex1, INFxcex2, INFxcex3, the pentapeptide pyroGlu-Glu-Asp-Cys-Lys, (SEQ ID NO:18) the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (SEQ ID NO:19), and the tripeptide glutathione (Gly-Cys-xcex3Glu).
The invention also includes a pharmaceutical composition comprising (a) INPROL and (b) at least one stimulatory compound selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-14, IL-15, G-CSF, GM-CSF, M-CSF, erythropoietin, thrombopoietin, stem cell factor, and flk2/flt3 ligand.
The current invention describes an inhibitory of stem cells (INPROL) which is different from those known in the art such as MIP-1xcex1, TGFxcex2, the tetrapeptide of Frindel and celleagues or the pentapeptide of Paukovits and coworkers (cf., Wright and Pragnell, 1992 (op cit)). Naturally occuring INPROL has a molecular weight exceeding 10,000 daltons by ultrafiltration which distinguishes it from the tetrapeptide as well as the pentapeptide. It is more hydrophobic than MIP-1xcex1 or TGFxcex2 in reverse phase chromatography systems, distinguishing it from those cytokines. Further, its mode of action is different from that of any previously described inhibitor in that it is active in an in vitro assay when used during a preincubation period only. MIP-1xcex1 for example, is not effective when used during a preincubation period only (Example 5). Further, naturally occuring INPROL is active in an assay measuring xe2x80x9chigh proliferative potential cellsxe2x80x9d (HPP-PFC) whereas MIP-1xcex1 is not (Example 6).